Zinc-sensing regulators enable cells to control Zn uptake, efflux, and storage that are critical for cell viability across all organisms. Many mechanistic pathways for these metalloregulators are still poorly understood. The long-term goal here is to understand how Zn homeostasis in the cell can be manipulated for preventative and therapeutic purposes. The overall objective here is to define how bacterial Zn-sensing metalloregulators (e.g., ZntR, Zur, and CzrA) ensure prompt switching between activation and deactivation and between repression and de-repression of their respective regulons in response to Zn excess or deficiency. The central hypothesis, formulated based on preliminary studies and past results, is that these metalloregulators use facilitated mechanisms for either protein dissociation from DNA or metal removal from the holo regulators in regulating transcription. To test this central hypothesis, the two PIs will collaborate and use the combined approach of single-molecule fluorescence imaging of protein-DNA interactions in vitro and in vivo, ensemble protein-DNA interaction kinetics, in vivo transcription profiling, chromatin immunoprecipitation, and protein and genetic engineering. The rationale for the proposed research is that the understanding of their regulation mechanisms will help in devising strategies and developing drugs to manipulate bacterial Zn uptake or efflux to limit their growth, thus contributing to the prevention and management of bacterial infectious diseases. The proposed research has three specific aims: (1) Identify the facilitated mechanisms of transcription deactivation by ZntR. The working hypotheses here are that the transcription deactivation of Zn efflux can occur via (#1) an assisted protein dissociation pathway, where free apo-ZntR, the repressor, assists the holo-ZntR, the activator, to dissociate from DNA, and/or (#2) a direct protein substitution pathway, where free apo-ZntR directly substitutes for the holo-ZntR on DNA. (2) Identify the facilitated mechanisms of transcription de-repression by Zur. The working hypotheses here are that (#3) free apo-Zur can assist holo-Zur, the repressor, to dissociate from DNA, leading to de-repression of Zn uptake, and (#4) bacillithiol (BSH) can facilitate Zn removal from DNA-bound holo-Zur to convert it to apo-Zur, which will dissociate from DNA leading to de-repression. (3) Identify the facilitated mechanisms of transcription de-repression and repression by CzrA. The working hypotheses here are that (#5) free holo-CzrA can assist apo-CzrA, the repressor, to dissociate from DNA, leading to de-repression of Zn efflux, and (#6) BSH can assist Zn removal from free holo-CzrA and accelerate subsequent CzrA binding to DNA for reestablishing repression. The research is significant because it will lead to the development of (bio)chemical strategies to manipulate bacterial Zn regulation to impair growth of pathogens and will fill knowledge gaps in Zn regulation mechanisms. The research is innovative because it integrates physical and biological sciences, combines both single-molecule and ensemble level measurements in vitro and in vivo, and introduces new concepts in transcription regulation.